The Gram positive bacterium Staphylococcus aureus is both an asymptomatic human colonizer and a pathogen that can cause infections in multiple tissue sites, including blood, skin and soft tissue, bone, and internal organs. Methicillin resistant Staphylococcus aureus (MRSA) is a common cause of death by hospital infections (HA-MRSA) and is now also a common community acquired infection (CA-MRSA). Vancomycin (a glycopeptide antibiotic) is the most commonly prescribed drug to treat MRSA infections. High-level resistance (minimal inhibitory concentration (MIC) ?16 ?g/ml) to vancomycin encoded by the mobile vanA gene is rare due to a fitness burden on S. aureus. However, it is more common to encounter strains with mutations conferring intermediate resistance to vancomycin arising from selection during the course of antibiotic therapy. The genetic basis of these vancomycin intermediate S. aureus (VISA) and heterogeneous resistant (hVISA) (MIC 2-8 ?g/ml) strains involves a large number of different genomic mutations that result in cell wall thickening through changes in cellular signaling and regulation. Routine phenotypic testing in clinical labs probably underestimates the incidence of VISA and hVISA. Due to the fact that mutations in several genes have been linked with VISA, genetic-based detection of intermediate vancomycin resistance has not been developed for routine clinical microbiological use. In our preliminary work, we created an extensive catalog of sequenced clinical and laboratory-selected VISA as well as databases of SNPs and genetic variation in thousands of public S. aureus genomes. In this work we plan to extend these studies toward development of a sequence-based testing protocol that could be used for large numbers of clinical strains. In Specific Aim 1 we plan to extend our knowledge of the mutations that cause VISA by sequencing a panel of 300 novel mutants strains spontaneously selected from 40 S. aureus parent genotypes. We estimate, based on the results of the preliminary data, that this number of strains will be sufficient identify mutations found in 95% of VISA strains. These data will be used for creation of a comprehensive VISA detection assay based on whole genome data with an accuracy of at least 95%. In Specific Aim 2 we will use the information learned from Aim 1 to create a multiplex PCR sequence test for VISA, VRSA and other resistance determinants of S. aureus based on the commercially available Fluidigm platform. We will ultimately aim to have an assay that can be used to monitor systemic MRSA infections, such as bacteremia, to detect development of VISA in its early stages in clinical specimens from the patient. The test will also be able to detect other S. aureus resistance phenotypes and call the genotype of the strain.